The long-range goal of this project is to further our understanding of how K+ channels work at a molecular level. The project focuses on the interaction between a Shaker K+ channel from Drosophila melanogaster, and charybdotoxin (CTX), a pore-blocking peptide inhibitor. CTX will be used to probe the external entryway to the conduction pore of this genetically manipulable channel, in order to identify and characterize this important part of the ion channel. Shaker K+ channels will be expressed in Xenopus oocytes and studied using electrophysiological techniques. CTX-binding regions will first be identified by producing site-directed mutant Shaker K+ channels that display altered CTX inhibition. A major effort will be put towards understanding the molecular mechanism by which a given mutation influences the interactions with toxin. For example, a particular glutamate on the channel has already been shown to influence the binding of the cationic CTX molecule by a simple through-space electrostatic mechanism; the glutamate must therefore be physically close to the CTX binding site. The next step in the project will be to ask if the amino acid residues which affect toxin binding also play a role in the ion conduction process. This will be determined by studying the single-channel current properties of the mutant Shaker K+ channels. The expectation is, based on preliminary results, that CTX will point out anionic residues in the channel's outer mouth which influence conducting ions by an electrostatic mechanism. This project will help to define the transmembrane orientation of the Shaker K+ channel (because the toxin blocks only from the outside), it will identify residues that make up the channels's external conduction pore entryway, and it may point out adjacent protein domains that form the deeper regions of the pore. The proposed project is health related; many cellular processes that are operating in virtually every organ system of the human body depend on K+ channels.